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Background Indocyanine green (ICG)-enhanced fluorescence optical imaging (FOI) is an established technology for imaging of inflammation in animal models. In experimental models of arthritis, FOI findings corresponded to histologically proven synovitis. This is the first comparative study of FOI with other imaging modalities in humans with arthritis. Methods 252 FOI examinations (Xiralite system, mivenion GmbH, Berlin, Germany; ICG bolus of 0.1 mg/kg/body weight, sequence of 360 images, one image per second) were compared with clinical examination (CE), ultrasonography (US) and MRI of patients with arthritis of the hands. Results In an FOI sequence, three phases could be distinguished (P1–P3). With MRI as reference, FOI had a sensitivity of 76% and a specificity of 54%, while the specificity of phase 1 was 94%. FOI had agreement rates up to 88% versus CE, 64% versus greyscale US, 88% versus power Doppler US and 83% versus MRI, depending on the compared phase and parameter. FOI showed a higher rate of positive results compared to CE, US and MRI. In individual patients, FOI correlated significantly (p

Impairment of the blood-brain barrier (BBB) after cerebral ischemia leads to extravasation of plasma constituents into the brain parenchyma. We describe a novel method using non-invasive near-infrared fluorescence (NIRF) imaging and bovine serum albumin labeled with a NIRF dye (NIRF-BSA) to detect BBB impairment after middle cerebral artery occlusion (MCAO) in mice. We first explored the time course of BBB impairment after transient MCAO using Evans blue (EB), which binds to plasma albumin in vivo. An initial BBB impairment was observed at 4-8h and a second impairment at 12-16h after reperfusion. No EB extravasation was detected at 8-12h. Non-invasive NIRF imaging with NIRF-BSA confirmed biphasic BBB impairment. Upon co-injection of NIRF-BSA with EB we found a strong correlation between the detected NIRF signal and the amount of extravasated EB (r=0.857, P=0.00178). When MCAO mice received NIRF-BSA together with gadolinium-diethylene triamine penta-acetic acid (Gd-DTPA), T1-weighted images showed Gd-DTPA enhancement at all times while NIRF imaging showed biphasic BBB impairment. In conclusion, NIRF-BSA is a suitable marker of plasma albumin extravasation in the mouse brain. Non-invasive NIRF imaging with NIRF-BSA is a useful tool to study BBB integrity in preclinical models of central nervous system pathology.

It has been shown that ED-B fibronectin (ED-B) is a potential target for plaque imaging. The aim of this study was to test a novel modified single chain anti-ED-B antibody (scFv) conjugated for near infrared fluorescence imaging (NIRF) with tetrasulfonated carbocyanine-maleimide (TSC-scFv) and to examine the association of ED-B with the presence of macrophages in a murine model of atherosclerosis. Expression of ED-B was observed in plaque areas in apolipoprotein E-deficient (apoE(-/-)) mice which increased with age and plaque load. Robust imaging was possible after explantation of the aorta and demonstrated a strong NIRF signal intensity in focal aortic and brachiocephalic plaque lesions, whereas no signals were found in undiseased areas. Plaque lesion ED-B was expressed by smooth muscle cell and was closely associated to macrophage infiltrates. Although not expressed by the same cell type, there was a significant correlation (p0.01) between ED-B and macrophage immunoreactivity. In vitro human coronary and mouse smooth muscle cells significantly increased ED-B expression after angiotensin II and TNF-alpha treatment. This study demonstrates that plaque NIRF imaging is feasible with a novel single chain antibody and that ED-B expression is closely associated with inflammation in experimental atherosclerosis.

The aim of the study is to evaluate in vivo fluorescence imaging of experimental inflammatory joint disease by applying two different near-infrared (NIR) dyes in a model of Borrelia-induced Lyme arthritis.Forty mice, 20 with Lyme arthritis and 20 controls, were examined. Two nonspecific NIR carbocyanine dyes, indocyanine green (ICG) and a hydrophilic carbocyanine derivative (1,1'-bis-[4-sulfobutyl] indotricarbocyanine-5,5'-dicarboxylic acid diglucamide monosodium salt [SIDAG]), were administered intravenously at two doses. Fluorescence images were acquired before and during 120 seconds after injection of cyanine dyes. For both dyes, the area under the curve (AUC) was determined for the interval between 40 and 80 seconds after injection. In addition, the slope of the signal decrease was compared among animal groups. Results were compared with histological findings.The general temporal fluorescence intensity course for ICG was characterized by a rapid increase, with a peak at 40-50 seconds followed by a decrease; conversely for SIDAG, by a slow increase. AUC analysis for both dyes showed that the fluorescence signal differed significantly between controls and arthritic animals (P.05). Within these groups, there were significant differences between the two doses investigated. ICG differed significantly between control and arthritic animals in the slope of the signal decrease for both doses investigated (P.05). Histological examination showed early stages of inflammation in arthritic animals.NIR fluorescence imaging based on the pharmacokinetic behavior of ICG or SIDAG is a promising approach to detect inflammatory joint changes of experimental arthritis. Moreover, SIDAG is suited to differentiate inflammatory and noninflammatory joints 24 hours after dye application.

To evaluate the feasibility of gene delivery mediated with diagnostic ultrasound and plasmid DNA (pDNA) encapsulated in gas-filled microparticles (GFMP) in rodent tumor models.This study was performed according to a protocol approved by the regional animal research committee. The model plasmid UT651 (pUT651) that contained the Escherichia coli LacZ gene for beta-galactosidase was used to demonstrate the feasibility of ultrasound-mediated gene delivery in CC531 liver tumors in rats. In preliminary experiments, a single injection of pUT651-containing GFMP was administered intraarterially (n=4) or intravenously (n=6) with simultaneous sonication (color Doppler mode, maximum mechanical index) of the GFMP passing through the capillaries of the tumors. All animals were sacrificed 2-5 days later, and liver tumors were examined for beta-galactosidase expression histochemically. Subsequently, potential medical usefulness of this delivery system was tested in nude mice bearing Capan-1 tumors (adenocarcinoma of the human pancreas) by using the plasmid RC/CMV-p16 (pRC/CMV-p16), which contains tumor suppressor gene p16. The tumor suppressor gene p16 is deleted in Capan-1 cells. Twenty-five tumor-bearing mice were classified into five groups (four to six mice per group, one treatment group, four control groups) at random. All mice were treated once weekly for 5 weeks with intravenous infusion of p16-containing GFMP or control substances with simultaneous tumor sonication with color Doppler mode ultrasound and maximum mechanical index or without ultrasound treatment. The therapeutic effect of p16 was measured as an increase in tumor volume doubling time. Data were analyzed with analysis of variance. Results were considered significant at the 5% critical level (P.05).A clear expression of pDNA was found in tumors in rats treated with a combination of pUT651-containing GFMP and ultrasound; relevant controls showed a significantly lower expression of marker gene. The controlled ultrasound-triggered release of pRC/CMV-p16 from GFMP leads to a strong tumor growth inhibition, which is significant (P.002), compared with that in controls.A combination of GFMP and ultrasound provides an effective approach for nonviral gene therapy-based cancer treatment.

To develop an in vitro assay for studying the feasibility of specific targeting of ultrasound contrast agents (USCAs) for ultrasound diagnostics by employing the parallel plate flow chamber, which provides an environment that mimics some aspects of the in vivo conditions like shear rate and flow effects.USCAs based on air-filled microparticles (MP) were functionalized with specific antibodies using carbodiimide coupling chemistry and characterized by fluorescence activated cell sorter (FACS). The binding experiments were done by subjecting the MP to shear stress as they interact with the target-coated surface of the flow chamber.A successive modification of MP with antibody and the glass surface with antigen was achieved and quantified. The binding studies showed specific attachment of targeted MP to EDB-FN (EDB domain of fibronectin) surface. The binding of MP via nonspecific interactions was minimal. The binding efficiency of antibody-loaded MP is dependent on the applied shear stress. An increase in the wall shear stress resulted in a decrease in binding efficiency. Binding efficiency was found to be correlated with the antibody density and antigen density on the interacting surfaces.The results indicate that the test system developed is reliable for characterizing targeted MP without any additional labeling and can be used as a functionality assay for studying the binding characteristic of USCA with respect to different parameters like density of targeting antibodies on the microparticle surface and of target protein. In addition, the microparticles can be studied in detail under different shear rates and flow conditions. Further studies concerning the in vitro-in vivo correlation will be necessary to further increase the value of this in vitro method.

To evaluate the feasibility of using intravenously administered L-selectin ligand-specific polymer-stabilized air-filled microparticles (MPs) for active targeting of peripheral lymph nodes under normal conditions in animal models.L-selectin ligand-specific MPs and two control substances (immunoglobulin M-isotype MPs and native MPs) were each administered in three conscious mice as a single intravenous bolus injection (1.4 x 10(7) MPs/kg). All mice were sacrificed 30 minutes after administration. Lymph nodes (cervical, inguinal, axillary, popliteal, mesenteric), spleen (positive control), and kidney (blood pool control) were removed and examined for MP-related stimulated acoustic emission (SAE) signals by using harmonic color Doppler ultrasonography (US) in a tank containing degassed water. A second experiment was performed in six anesthetized beagle dogs by using the same MP formulation. Each of the MP formulations was administered in two anesthetized dogs as a single intravenous bolus injection (1 x 10(7) MPs/kg). The popliteal lymph nodes, spleen (positive control), and kidney (blood pool control) were examined in vivo with US for MP-related SAE signals 30 minutes after administration. Fisher exact test for the one-side alternative was used for mouse data analysis.The lymph nodes of all mice (P =.05) and the popliteal lymph nodes of both dogs treated with L-selectin ligand-specific MPs showed clear MP-related SAE signals, whereas the lymph nodes of all mice and the popliteal lymph nodes of four dogs that received the control substances did not show any SAE signals.Use of an intravenously administered L-selectin ligand-specific US contrast agent is feasible for active lymph node targeting in mice and dogs.

Vascular endothelial growth factor (VEGF) is a potent mitogen with a unique specificity for endothelial cells and a key mediator of aberrant endothelial cell proliferation and vascular permeability in a variety of human pathological situations, such as tumor angiogenesis, diabetic retinopathy, rheumatoid arthritis, or psoriasis. VEGF is a symmetric homodimeric molecule with two receptor binding interfaces lying on each pole of the molecule. Herein we report on the construction and recombinant expression of an asymmetric heterodimeric VEGF variant with an intact receptor binding interface at one pole and a mutant receptor binding interface at the second pole of the dimer. This VEGF variant binds to VEGF receptors but fails to induce receptor activation. In competition experiments, the heterodimeric VEGF variant antagonizes VEGF-stimulated receptor autophosphorylation and proliferation of endothelial cells. A 15-fold excess of the heterodimer was sufficient to inhibit VEGF-stimulated endothelial cell proliferation by 50%, and a 100-fold excess resulted in an almost complete inhibition. By using a rational approach that is based on the structure of VEGF, we have shown the feasibility to construct a VEGF variant that acts as an VEGF antagonist.

Stable prostacyclin analogues exert a strong inhibitory effect on lymphogenous as well as haematogenous tumor metastasis in a series of tumor lines. The strong inhibition of metastasis was achieved by repeated once-daily i.g. applications. The mechanism of antimetastatic action is related to the expression of functional IP-receptors (PGI-receptors). As cellular assay systems indicated that the IP-receptor mediated signalling is down-regulated upon continuous exposure to prostacyclin or stable derivatives, it has been questioned whether a mode of drug application with constant plasma drug levels may potentially result in a decrease of the antimetastatic effect. We addressed this question using the stable prostacyclin analogue cicaprost in a disease model by comparing i.g. applications given once daily with a continuous administration of equivalent doses via drinking water. Very similar to our previous investigations in the 13762NF MTLn3 rat mammary carcinoma model, cicaprost administered by i.g. application strongly reduced lung and lymph node metastasis. In contrast, administration of equivalent doses via drinking water leading to lower but constant steady-state plasma levels failed to exert inhibitory effects. Plasma and urine levels of cicaprost were measured with a sensitive radioimmunoassay on the last treatment day. Pharmacokinetic evaluation demonstrated a similar bioavailability of cicaprost in both groups. This result first demonstrates a treatment failure of a prostacyclin derivative in a chronic disease model in association with a continuous drug administration leading to constant plasma levels. A desensitization of receptor signalling by constant plasma levels may be a possible mechanism for treatment failure.

In breast cancer, the survival rate strongly depends on the number of lymph nodes involved. A drug with a specific inhibitory activity on lymph node and organ metastases would therefore be a candidate for adjuvant therapy after surgery. Prostacyclin and its stable analogues have been shown to interfere with certain steps of the metastatic cascade and to inhibit the number of lung colonies after i.v.-inoculation of various tumor cell lines. Our data reveal that cicaprost, a metabolically stable and orally active analogue of prostacyclin, has pronounced antimetastatic effects in a series of spontaneously metastasizing rodent tumors. In the SMT 2a and 13762 MTLn3 mammary carcinomas of the rat, cicaprost given daily from the day of tumor implantation strongly inhibits the number of lung metastases as well as lymph node weights without exerting an effect on the primary tumor. Even starting treatment when palpable primary tumors are present gives a pronounced antimetastatic activity. To demonstrate that cicaprost has an effect on metastases already settled in the respective organ, treatment was started after surgical removal of the primary tumor. In the SMT 2a tumor, a strong inhibition of the number of metastases was shown. Interestingly, a perioperative treatment schedule was also effective in both models used. As primary tumor growth in vivo or proliferation in vitro remained unchanged by cicaprost, its mode of action seems to be related to one or more mechanisms of the metastatic process. In tumor cell lines expressing a functional prostacyclin receptor, stimulated tumor cell migration is inhibited and changes of differentiation status are obvious. In conclusion, cicaprost strongly inhibits lymph node and organ metastases of spontaneously metastasizing rodent mammary tumors with a mode of action different from cytostatic or antihormonal drugs.

Using 15 rats with collagen-induced arthritis (30 joints) and 7 control rats (14 joints), we correlated the intensity of near-infrared fluorescence (NIRF) of the nonspecific dye tetrasulfocyanine (TSC) with magnetic resonance imaging (MRI), histopathology, and clinical score. Fluorescence images were obtained in reflection geometry using a NIRF camera system. Normalized fluorescence intensity (INF) was determined after intravenous dye administration on different time points up to 120 min. Contrast-enhanced MRI using gadodiamide was performed after NIRF imaging. Analyses were performed in a blinded fashion. Histopathological and clinical scores were determined for each ankle joint. INF of moderate and high-grade arthritic joints were significantly higher (p

More than a decade ago, prostacyclin, a dienoic bicyclic eicosanoid derived from the metabolism of arachidnoic acid, was found to possess potent inhibitory effects on tumor cell metastasis. Thereafter, several laboratories demonstrated the metastasis-suppressive activity of prostacyclin in a wide spectrum of tumor types. Due to the short half-life of prostacyclin, researchers have focused on looking for stable prostacyclin analogues which have extended half lives and increased bioavailabilities. Cicaprost, among other prostacyclin analogues tested, has been demonstrated, like prostacyclin, to effectively inhibit metastasis in several different animal models (i.e., both experimental and spontaneous metastasis models). Prostacyclin as well as cicaprost prevent not only hematogenous, but also lymphatic metastasis. Furthermore, these compounds also inhibit the growth of established micrometastases after removal of the primary tumors. Mechanistic studies revealed that the antimetastatic effects of prostacyclin and its analogues are more related to their interference with tumor cell-host interactions (such as tumor cell induced platelet aggregation, tumor cell adhesion to endothelial cells and subendothelial matrix, tumor cell induced endothelial cell retraction, etc.) than their direct inhibition of the growth of primary tumors. The potent and widespread metastasis-retarding effects of prostacyclin and its stable analogues in animal tumor models warrant their clinical trial in treating human cancer patients and preventing metastasis.

Prostacyclin and its stable analogues have been shown to interfere specifically with certain steps of the metastatic cascade. The antimetastatic activity of the stable prostacyclin analogue Cicaprost (Schering AG) on haematogenous metastasis in a series of tumours in rats and mice has been well established. In order to test the effect of Cicaprost on lymphogenous metastasis we chose the metastatic cell clone MTLn3 derived from the 13762NF rat mammary carcinoma. The effect of Cicaprost on prevention of lung metastasis, lymph node metastasis and primary tumour growth was investigated. Cicaprost given in daily doses of 0.01, 0.03 and 0.1 mg/kg orally, reduced the number of lung metastases in a dose-dependent manner. Whereas the median number of lung metastases in the controls was greater than 1000, Cicaprost at a dose of 0.1 mg/kg reduced the number of lung metastases to between 11 and 100. The weight of the ipsilateral axillary lymph nodes was diminished by Cicaprost to 30-50% of controls. Moreover, metastasis to the contralateral axillary lymph node was completely inhibited by Cicaprost at all three doses tested. Cicaprost did not influence the growth rate of the MTLn3 cell clone implanted into the mammary fat pad or the weight of the primary tumour at the end of treatment. In conclusion, in addition to its dose-dependent effect on haematogenous metastasis, Cicaprost strongly inhibits lymph node metastasis.

Background According to the World Health Organization (WHO) there is a pandemic of obesity with approximately 300 million people being obese. Typically, human obesity has a polygenetic causation. Neutral endopeptidase (NEP), also known as neprilysin, is considered to be one of the key enzymes in the metabolism of many active peptide hormones. Methodology/Principal Findings An incidental observation in NEP-deficient mice was a late-onset excessive gain in body weight exclusively from a ubiquitous accumulation of fat tissue. In accord with polygenetic human obesity, mice were characterized by deregulation of lipid metabolism, higher blood glucose levels, with impaired glucose tolerance. The key role of NEP in determining body mass was confirmed by the use of the NEP inhibitor candoxatril in wild-type mice that increased body weight due to increased food intake. This is a peripheral and not a central NEP action on the switch for appetite control, since candoxatril cannot cross the blood-brain barrier. Furthermore, we demonstrated that inhibition of NEP in mice with cachexia delayed rapid body weight loss. Thus, lack in NEP activity, genetically or pharmacologically, leads to a gain in body fat. Conclusions/Significance In the present study, we have identified NEP to be a crucial player in the development of obesity. NEP-deficient mice start to become obese under a normocaloric diet in an age of 6–7 months and thus are an ideal model for the typical human late-onset obesity. Therefore, the described obesity model is an ideal tool for research on development, molecular mechanisms, diagnosis, and therapy of the pandemic obesity.

The blood pool agent indo-cyanine green (ICG) has been investigated in a prospective clinical study for detection of rheumatoid arthritis using fluorescence imaging. Temporal behavior as well as spatial distribution of fluorescence intensity are suited to differentiate healthy and inflamed finger joints after i.v. injection of an ICG bolus.

The dye ICG was investigated in a clinical study for evaluation of rheumatoid arthritis using fluorescence imaging. Second moments of correlated time series of fluorescence intensities were analysed to differentiate healthy subjects from diseased.

We investigated a molecular imaging approach for the detection of collagen-induced arthritis in rats by targeting the extra domain B (ED-B) of the extracellular matrix protein fibronectin. ED-B is a highly conserved domain (identical in human and rats) that is produced by alternative splicing during embryonic development and during vascular remodeling such as angiogenesis. The hallmark of rheumatoid arthritis is synovitis leading to both angiogenesis in the synovium and the promotion of cartilage and bone disruption. For in vivo diagnostics, the ED-B-binding single-chain antibody fragment AP39 was used as a targeting probe. It was covalently linked to the near-infrared dye tetrasulfocyanine (TSC) to be visualized by near-infrared fluorescence imaging. The resulting AP39-TSC conjugate was intravenously administered to rats with collagen-induced arthritis and the respective controls. Ovalbumin-TSC was used as control conjugate. Optical imaging over a time period of 24 hours using a planar imaging setup resulted in a clear enhancement of fluorescence intensity in joints with moderate to severe arthritis compared with control joints between 3 and 8 hours postinjection. Given that AP39 is a fully human antibody fragment, this molecular imaging approach for arthritis detection might be translated to humans.

Introduction Use of fluorescence imaging in oncology is evolving rapidly, and nontargeted fluorochromes are currently being investigated for clinical application. Here, we investigated whether the degree of tumour angiogenesis can be assessed in vivo by planar and tomographic methods using the perfusion-type cyanine dye SIDAG (1,1'-bis- [4-sulfobutyl]indotricarbocyanine-5,5'-dicarboxylic acid diglucamide monosodium). Method Mice were xenografted with moderately (MCF7, DU4475) or highly vascularized (HT1080, MDA-MB435) tumours and scanned up to 24 hours after intravenous SIDAG injection using fluorescence reflectance imaging. Contrast-to-noise ratio was calculated for all tumours, and fluorochrome accumulation was quantified using fluorescence-mediated tomography. The vascular volume fraction of the xenografts, serving as a surrogate marker for angiogenesis, was measured using magnetic resonance imaging, and blood vessel profile (BVP) density and vascular endothelial growth factor expression were determined. Results SIDAG accumulation correlated well with angiogenic burden, with maximum contrast to noise ratio for MDA-MB435 (P < 0.0001), followed by HT1080, MCF7 and DU4475 tumours. Fluorescence-mediated tomography revealed 4.6-fold higher fluorochrome concentrations in MDA-MB435 than in DU4475 tumours (229 ± 90 nmol/l versus 49 ± 22 nmol/l; P < 0.05). The vascular volume fraction was 4.5-fold (3.58 ± 0.9% versus 0.8 ± 0.53%; P < 0.01), blood vessel profile density 5-fold (399 ± 36 BVPs/mm2 versus 78 ± 16 BVPs/mm2) and vascular endothelial growth factor expression 4-fold higher for MDA-MB435 than for DU4475 tumours. Conclusion Our data suggest that perfusion-type cyanine dyes allow assessment of angiogenesis in vivo using planar or tomographic imaging technology. They may thus facilitate characterization of solid tumours.

The blood pool agent indo-cyanine green (ICG) has been investigated for early detection of rheumatoid arthritis using fluorescence imaging. Different distributions of fluorescence intensity are found for healthy and inflamed finger joints after i.v. injection of an ICG bolus .

Background: Purpose of the study was to detect early breast cancer and its precursors by in vivo optical molecular imaging in an animal model. Material and methods: Females of the transgene mouse strain WAP-TNP8 develop non-invasive and consequently invasive tumours of the mammary glands due to specific expression of the viral SV40 large tumour antigen induced by lactation. The molecular target for imaging was extradomain-b fibronectin (EDB-FN), which is associated with tumour angiogenesis. The optical probe was designed as a compound of an anti-EDB-FN antibody fragment and a near-infrared fluorescent dye. 30h after intravenous injection of the contrast agent, optical imaging was performed using a pulsed Laser system for excitation and an intensified CCD-camera to record fluorescence images. After optical molecular imaging all animals were sacrified and the tumours were examined histologically. Results: Initiated transgene female animals developed palpable masses of the mammary gland within 6 months (median 4 months). Imaging was performed in 5 animals with a total of 9 tumours (diameter 2–7mm, median 4mm). Applying optical molecular imaging 8 of 9 tumours were detected. The urogenital tract was contrasted unspecifically. Histological examination proved invasive epithelial tumours of the mammary gland in all cases. Conclusion: Breast cancer can be detected in vivo by near-infrared fluorescence molecular imaging targeting neoangiogenesis in a transgene mouse-model.

The infiltration of autoreactive T cells into the central nervous system (CNS) requires a complex molecular interplay between immune cells and the blood-brain barrier (BBB), especially involving adhesion molecules like intercellular adhesion molecule (ICAM)-1. Here we study the molecular expression at the BBB during adoptively transferred (AT) myelin basic protein (MBP)-experimental autoimmune encephalomyelitis (EAE) in vivo by sensitive particle acoustic quantification (SPAQ)-enhanced ultrasound after intravenous application of specific gas-filled MP (MP) targeted against ICAM-1 (ICAM-MP) as contrast agent. Our results reveal a clear periventricular and cerebellar upregulation of ICAM-1 expression at the disease maximum of AT-EAE. Moreover. SPAQ-enhanced ultrasound enables the sensitive quantification of ICAM-1 expression in vivo. This allows to monitor therapeutic changes as shown by suppression of ICAM-1 expression after pretreatment of rats with corticosteroids (P < 0.008). All imaging results were confirmed by parallel immunohistochemistry. In vivo magnetic resonance imaging and albumin staining of rat brains after sonification did not reveal a disturbance of the BBB, thereby proving the safety of the method. Subsequent application of specific MP did not influence follow-up measurements, a prerequisite for sequential measurements in longitudinal studies. Based on these data, quantitative molecular imaging of adhesion molecules by SPAQ-enhanced ultrasound proves to be a safe and reliable technology to monitor changes at the BBB in vivo.

Molecular imaging requires, not only the identification of an appropriate marker, but also its quantitative analysis. We used the Sensitive Particle Acoustic Quantification (SPAQ) technology - a novel ultrasound technique - for detection and quantification of cell adhesion molecules in isolated tissue and in live animals. By conjugating gas-filled microparticles (MPs) with antibodies to intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), we were able to depict and quantify ICAM-1 and VCAM-1 in isolated brain and spinal cord from rats with autoimmune encephalomyelitis (EAE), an established inflammatory disease model of human multiple sclerosis (MS). Depiction and quantification of specific MPs were also feasible in living animals with AT-EAE with similar results. After treatment with methylprednisolone, the measured number of targeted anti-ICAM-1 and VCAM-1-MPs was significantly lower (P0.01) compared to untreated animals demonstrating the high sensitivity of this imaging technique. Depending on the antibody linked to the surface of the MPs, the technique can be used to quantify the expression of any accessible antigen expressed on the luminal surface of endothelial cells and is therefore a promising tool for the non-invasive and dynamic assessment of disease-related molecules.

Advances in imaging provide new insights into the pathophysiol-ogy of many diseases. Established imaging technologies such as MRI, CT, PET, and ultrasound are routinely applied to determine features of tumor blood vessels that distinguish them from normal blood vessels. These techniques yield information on blood flow, blood volume, and vessel permeability. Often, an intravenously injected imaging contrast agent without affinity to a specific target structure is applied to enable detection of malignant lesions. One of the emerging innovations in diagnostic imaging is the evolution of molecular imaging techniques. Molecular imaging is a noninvasive approach to determine the expression of indicative marker molecules of the tumor angiogenesis process. Meanwhile, this approach has been established for all imaging modalities and may further improve sensitivity of diagnostic tumor imaging. Another goal is to provide information with respect to drug treatment monitoring and therapeutic vascular targeting strategies.

Fluorescence imaging in the near-infrared range following intravenous administration of contrast agents has been investigated as a new tool for detection of inflammatory joint diseases. Our study reveals that NIR dyes accumulate in inflammatory joints.

Angiotensin II (ANG II), a key regulator of blood pressure and body fluid homeostasis, exerts mitogenic effects on endothelial cells. We therefore hypothesized that ANG II could be a mediator between homeostatic changes within the vascular perfusion bed and growth factor-driven angiogenesis. In the present study, we applied the alginate implant angiogenesis model in mice with normal ANG II levels, elevated ANG II levels by transgenic overexpression of angiotensinogen (AOGEN), or in AT2 receptor-deficient mice. We demonstrate that a decrease in the amount of circulating ANG II by the angiotensin-converting enzyme (ACE) inhibitor enalapril or the AT1 receptor antagonist losartan induced a stimulation of in vivo angiogenesis implying an inhibitory function of ANG II through the AT1 receptor. However, the strong increase of angiogenesis in AOGEN-transgenic mice compared with mice with normal ANG II levels suggests additional stimulatory activity. We showed that the ANG II-induced stimulation of angiogenesis is linked to the AT2 receptor as an impaired induction of angiogenesis was obtained in AT2 receptor knockout mice. These findings provide the first evidence that the AT2 receptor mediates a stimulation of in vivo angiogenesis and indicate that ANG II is a humoral regulator of peripheral angiogenesis involving two receptor subtypes with opposing actions.

Interactions of tumor cells with lymphatic vessels are of paramount importance for tumor progression, however, the underlying molecular mechanisms are poorly understood. Whereas enlarged lymphatic vessels are frequently observed at the periphery of malignant melanomas, it has remained unclear whether intratumoral lymphangiogenesis occurs within these tumors. Here, we demonstrate the presence of intratumoral lymphatics and enlargement of lymphatic vessels at the tumor periphery in vascular endothelial growth factor (VEGF)-C-overexpressing human melanomas transplanted onto nude mice. VEGF-C expression also resulted in enhanced tumor angiogenesis, indicating a coordinated regulation of lymphangiogenesis and angiogenesis in melanoma progression. The specific biological effects of VEGF-C were critically dependent on its proteolytic processing in vivo. Furthermore, VEGF-C induced chemotaxis of macrophages in vitro and in vivo, revealing a potential function of VEGF-C as an immunomodulator. Taken together, our results identify VEGF-C as multifunctional factor involved in regulating tumor lymphangiogenesis, angiogenesis, and immune response.

Background Fluorescence optical imaging (FOI) is a novel imaging technology. It detects hypervascularisation of inflamed joints and tissues in both hands using indocyanine green (ICG) as fluorophore. Objectives In this study, FOI was compared to low-field MRI and musculoskeletal ultrasound (US) in patients with early rheumatoid arthritis (RA). Methods Thirteen patients (9 female; mean age 54±15 yrs, range 26-75) with the diagnosis of early RA were enrolled. Low-field MRI (under application of 0.2 mmol/kg BW Dotarem®) and US (grey-scale (GSUS) as well as power-Doppler (PDUS) mode) of the clinically more affected hand were performed. Both methods were evaluated according to the OMERACT criteria. After informed consent, all patients were examined by the FOI device Xiralite© (mivenion GmbH, Berlin) using ICG as fluorophore (ICG-Pulsion® 0.1 mg/kg BW bolus I.V., 6 minutes examination time). Images were interpreted in three defined phases of increased signal intensity (ISI) in the finger tips: early (P1, prior to strong ISI in the finger tips), intermediate (P2, during ISI in the finger tips), and late phase (P3, after decreasing of ISI in the finger tips) [1]. Sensitivity and specificity for synovitis were calculated using MRI as reference. Furthermore, interclass correlation coefficients (ICC) were calculated between all three imaging methods. Results In total, 104 joints (MCP n=52, PIP n=52) were evaluated. For all finger joints, FOI reached a sensitivity of 88% and specificity of 57% using MRI as reference. In detail, sensitivity of 70% and specificity of 81% was calculated for the MCPs (n=52), and sensitivity of 96% and specificity of 41% for the PIPs (n=52) (Table 1). The ICCs between MRI and FOI were 0.77 for the MCPs and 0.79 for the PIPs. The ICCs between US and FOI were 0.79 for the MCPs and 0.73 for the PIPs (Table 2). Conclusions With MRI as reference, the novel FOI method seems to be very sensitive in the detection of synovitis, but not very specific. There was a high sensitivity especially for the PIP joint evaluation, with poor specificity, though. In contrast, MCP joint evaluation by FOI was much more specific. There were comparable good correlations between FOI and MRI as well as between FOI and US. Hence, FOI might be a helpful tool in the screening of patients with suspected early RA. References Werner SG, Langer EH, Ohrndorf S, et al. Inflammation assessment in patients with arthritis using a novel in vivo fluorescence optical imaging technology.Ann Rheum Dis. 2011 Oct 12. [Epub ahead of print] Disclosure of Interest S. Ohrndorf Grant/Research support from: Arthromark Research Support, M. Krohn: None Declared, S. Werner Grant/Research support from: Pfizer Research Support, M. Bahner: None Declared, M. Schirner: None Declared, B. Reiche: None Declared, S. Schneider: None Declared, L. Braum: None Declared, G. Burmester: None Declared, B. Hamm: None Declared, K.-G. Hermann: None Declared, M. Backhaus Grant/Research support from: Pfizer Research Support

Background Assessment of arthritis using fluorescence optical imaging (FOI) in combination with the fluorophore indocyanine green (ICG) is now clinically established [1]. Beyond imaging of inflammation, FOI also provides the opportunity to monitor drug targeting of biological therapeutics into inflamed joints. Quantitative information of drug targeting would allow for patient selection and individual treatment schedules. Objectives We report the first synthesis of etanercept-ICG-conjugates and the quantitative monitoring of drug targeting into inflamed joints of collagen II induced arthritis using a modified fluorescent camera system (Xiralite). Methods Inflammatory arthritis was induced by twice injections of bovine collagen II (day 0, day 7) in female Lewis rats. Rats were monitored for joint swelling, pain, redness and mobility (score 0 to 3). The TNFa binding protein etanercept (Enbrel®, Pfizer) was chemically conjugated with an ICG derivative suited for labeling of proteins. The etanercept-6S-ICG (Eta-6S-ICG) conjugate was photophysically characterized and tested for binding affinity The fluorescence camera system Xiralite (mivenion GmbH, Germany) [1] was modified to enable imaging of rats. On day 14 after first injection of collagen, Eta-6S-ICG (n=6), IgG-6S-ICG (n=3), or the inflammation-targeted macromolecule polyglycerolsulfate-6S-ICG (dPGS-6S-ICG, n=5) [2] were s.c. or i.v. injected in a dose of 10 nMol/kg b.w. Fluorescence measurement was performed before and 1 h, 3 h, 24 h and 48 h after injection. Fluorescence intensity (FI) was measured in ROI over the tibio-tarsal and interphalangeal articulations and compared with healthy reference ROI. In addition, targeting was studied in rat tissues 3 or 24 h post injection of Eta-6S-, IgG-6S- or dPGS-6S-indocarbocyanine conjugates using fluorescence histology. Results Binding affinity of etanercept was not attenuated by chemical conjugation with 6S-ICG. Eta-6S-ICG exerts high fluorescence quantum yield compared with ICG. FI was detected already 24 hours after administration of Eta-6S-ICG and further increased until 48 hours after injection. Significant FI at 48 hours after injection of Eta-6S-ICG was measured in tibio-tarsal joints (score 1: 15.8±2.9; score 3: 31.0±4.6) while FI in interphalangeal joints was low (score 1: 6.3±1.0; score 3: 7.7±1.2). In contrast, dPGS-6S-ICG exhibited high FI in both tibio-tarsal joints (score 1: 23.5±4.9; score 3: 69.8±17.5) and interphalangeal joints (score 1: 20.9±5.8; score 3: 37.0±4.7). Both Eta-6S- ICG and dPGS-6S-ICG show strong targeting to inflamed synovial tissue and cartilage compared with IgG-6S-ICG. Conclusions FOI and labeling of macromolecular therapeutics with novel ICG derivatives provide a novel rationale for sensitive and quantitative monitoring of arthritis therapy. Further studies have to determine the effect of varying molecular size on the degree of targeting of inflamed joints. References Werner S et al. Inflammation assessment in patients with arthritis using a novel in vivo fluorescence optical imaging technology. Ann Rheum Dis doi:10.1136/ard.2010.148288. Licha K et al. Fluorescence imaging with multifunctional polyglycerol sulfates: novel polymeric near-IR probes targeting inflammation. Bioconjug Chem; doi:10.1021/bc2002727 Disclosure of Interest P. Welker Employee of: mivenion GmbH, K. Licha Shareholder of: mivenion GmbH, I. Gemeinhardt: None Declared, M. Bahner Shareholder of: mivenion GmbH, J. Berger Employee of: mivenion GmbH, D. Mangoldt Employee of: mivenion GmbH, J. Schnorr: None Declared, M. Schirner Shareholder of: mivenion GmbH

Background Diagnostic imaging, including ultrasound and MRI is routinely used in joint diseases. Besides initial diagnosis of the disease, imaging is also of strong interest for monitoring of treatment response. In this regard, semi-quantitative scores, like the RAMRIS or the US7 – score, aim at measuring disease activity. Most recently, fluorescence optical imaging (FOI) with the Xiralite® system became clinically available for diagnostic imaging of inflammation in the hands [1]. So far, a quantitative method for measuring disease activity has not been published. Objectives We first report a method for an automated, computer-based algorithm for real-time, quantitative analysis of FOI images. Methods 15 FOI standard exams were randomly selected from the multicenter OPERA1 trial (rheumatoid arthritis = 7; psoriatic arthritis = 8). These and 6 exams from normal individuals without clinical symptoms were calculated. Composite images of the first 240 seconds were generated. Using dedicated parameters of the histograms, automatic extraction of left and right hands from the image background was performed. After the extraction, the investigational algorithm separated the hands from the forearm in a standardized manner by using a multiple of the length of the middle finger. Next, a threshold in the fluorescence intensity curve was set to discriminate and measure high and low intensity areas in patients and healthy individuals. Next, high intensity areas of patients were compared with the mean value of normal individuals which was defined as reference value. Disease activity (DACT) is expressed as signal intensity (SI) (threshold to max patient/mean SI (threshold to max)healthy. DACT in healthy individual equals 1 which relates to an area of high intensity of 3.6%. Results 21 left and 21 right hands were correctly extracted from the auto-scaled composite image of the first 240 seconds. The intra-individual variation of the extraction procedure measured as high intensity area in three consecutive procedures was Conclusions Automatic, computer-based image analysis of FOI using dedicated software is technically feasible and offers high reproducibility. Measurement of areas of high signal intensity may provide a reliable quantitative readout for inflammatory activity. Varying doses of ICG do not have an obvious impact on auto-scaled composite images or calculated values. Further clinical investigations have to determine the agreement with other clinical measures (e.g. DAS28, ACR). References Werner SG et al. Inflammation assessment in patients with arthritis using a novel in vivo fluorescence optical imaging technology. Ann Rheum Dis 2011: in press Disclosure of Interest M. Schirner Shareholder of: mivenion, Employee of: mivenion, M. Cziumplik Employee of: mivenion, M. Bahner Shareholder of: mivenion, Employee of: mivenion, J. Berger Employee of: mivenion

Background Fluorescence optical imaging (FOI) with the Xiralite® system is clinically available for diagnostic imaging of inflammation and has been shown to be a suitable tool to assess treatment response in subjects with RA and PsA. Changes in FOI are measured using the semi-quantitative fluorescence optical imaging activity score (FOIAS) which allows for a low inter-reader variability 1 . Most recently, an automatic, computer-based image analysis of FOI (DACT) has been described and validated in healthy subjects 2 . Objectives To evaluate an automatic computer-based analysis of FOI in patients with RA or PsA under treatment. Methods 30 patients with rheumatoid (RA) or psoriatic (PsA) arthritis were examined before starting treatment (visit 1) and at follow-up (visit 2). Treatment response was assessed using DAS28, cFOIAS and the automated calculated DACT. First, 3 readers analyzed all 60 image data sets individually. For articular location of increased signal intensities (ISI) the FOIAS was used. For extraarticular location the signals were graded semiquantitatively on a scale of 0 to 2. These values were added to a single number representing the overall fluorescence signal intensity of the hands (cFOIAS). For DACT composite images were generated automatically. Using dedicated parameters of the histograms, automatic extraction of the hands from the image background was performed by the computer. The algorithm separated the hands from the forearm in a standardized manner by using a multiple of the length of the middle finger. A threshold in the fluorescence intensity curve was set to discriminate high and low intensity areas. The area of high intensity was calculated and the values of each patient were divided by the 95th percentile of normal individuals which was defined as reference value. DACT was expressed as high signal intensity area patient / healthy reference. DACT in healthy individuals equals 1. Then cFOIAS and DACT values were compared. Results 60 left and 60 right hands were correctly extracted from the auto-scaled composite image of the first 240 seconds. The intra-individual variation of the extraction procedure measured as intensity area of the hand in the consecutive procedures was 1.1 % + 1.0 (range: 0.04 – 4.9). cFOIAS scores strongly correlate with automated calculated DACT (0.86 / reader 1, 0.84 / reader 2 and 0.85 / reader 3). Agreement of declining DAS28 with declining cFOIAS was found in 16/30 (reader 1), 17/30 (reader 2) and 15/30 (reader 3) treatment courses. Agreement of declining DAS28 with declining DACT was found in 16/30 treatment course. Conclusions This first clinical trial in patients with RA or PsA under treatment with follow-up FOI exams demonstrates that automatic, computer-based image analysis of FOI to calculate DACT values is technically feasible, offers high reproducibility and high agreement with cFOIAS score reading, while agreement of DACT value with DAS28 was only moderate. Even very low changes of the DACT value can be measured and considered as statistically significant. References Werner SG et al. Indocyanine Green Enhanced Optical Imaging Using for Monitoring of Treatment Response. Arthritis & Rheumatism 2011: 63 (10); S73 Schirner M. et al. Methods for automated, computer-based quantitative assessment of fluorescence optical imaging. Ann Rheum Dis 2012;71(Suppl3):711 Disclosure of Interest M. Bahner Shareholder of: mivenion GmbH, Employee of: mivenion GmbH, S. Werner: None Declared, M. Cziumplik Employee of: mivenion GmbH, J. Berger Employee of: mivenion GmbH, H.-E. Langer: None Declared, M. Schirner Shareholder of: mivenion GmbH, Employee of: mivenion GmbH

Fluorescence imaging using the dye indocyanine green as a contrast agent was investigated in a prospective clinical study for the detection of rheumatoid arthritis. Normalized variances of correlated time series of fluorescence intensities describing the bolus kinetics of the contrast agent in certain regions of interest were analyzed to differentiate healthy from inflamed finger joints. These values are determined using a robust, parameter-free algorithm. We found that the normalized variance of correlation functions improves the differentiation between healthy joints of volunteers and joints with rheumatoid arthritis of patients by about 10% compared to, e.g., ratios of areas under the curves of raw data.

Spontaneously hypertensive rats (SHR) of the Okamoto strain with blood pressure above 161 mmHg and SHR with blood pressure levels of less than 160 mmHg were treated with oral doses of aspirin (100 mg kg-1) for three days. Whereas the blood pressure of SHR with blood pressure above 161 mmHg was decreased by aspirin, the blood pressure of SHR below 160 mmHg was increased by aspirin. The extent and direction of blood pressure change by aspirin was strongly correlated with the blood pressure of SHR before treatment (r = -0.88). The effect of aspirin supports an important role for endogenous prostanoids in the regulation of blood pressure of SHR.

We investigated the effect of cisplatin given at different treatment intervals on growth of the s.c.-implanted M 5076 reticulum sarcoma and the number of liver metastases at the end of the experiment (day 24). The later the treatment was started, the smaller was the tumour-inhibiting effect of cisplatin on primary tumour growth. Initiation of cisplatin treatment before day 14 after tumour implantation inhibited liver metastases completely. Treatment starting from day 14 or later did not influence the number of liver metastases. With regard to the clinical situation, the data imply that in the search for new leads in anticancer compounds, experimental conditions should concentrate on the inhibition of metastatic tumour growth.

Investigations on mechanisms of metastatic tumour spread revealed a role for compounds that inhibit tumour dissemination at the time of hematogenous dissemination. The platelet aggregation inhibitor prostacyclin and its stable analogues were shown to inhibit tumour-cell-induced platelet interaction as well as tumour cell adhesive mechanisms. This study concentrates on the effect of the stable prostacyclin analogue cicaprost: 5-{(E)-(1S, 5S, 6S, 7R)-7-hydroxy-6-[(3S, 4S)-3-hydroxy-4-methylnona-1,6-diinyl]-bicyclo[3,3,0]octan-3-ylidene}-3-oxapentanoic acid (Schering AG), as cyclodextrin clathrate, on spontaneous tumour metastases of two different carcinomas of the rat. In Cop rats bearing spontaneously metastasizing R 3327 MAT Lu prostate carcinomas, cicaprost (1.0 mg/kg p.o. daily) inhibited the number of lung metastases by about 80%, whereas the lower doses (0.1 and 0.5 mg/kg) exhibited borderline efficacy. In female Wistar-Furth rats bearing s.c. implanted SMT 2A mammary carcinomas, spontaneously metastasizing into regional lymph nodes and lungs, cicaprost (0.1, 0.5 and 1 mg/kg) p.o. daily exhibited a dose-dependent inhibition of the number of lung metastases. Five out of ten animals treated by 1 mg/kg were free of visible lung metastases. The weight of the axillary lymph node was significantly reduced by the 1 mg/kg dose of cicaprost, whereas lower doses has no effect on the weight of the lymph nodes. The growth of the primary tumour was not influenced by cicaprost in the R 3327 MAT Lu prostate carcinoma nor in the SMT 2A mammary carcinoma in the dose range tested. In conclusion, the stable prostacyclin analogue cicaprost exhibits a strong antimetastatic action in two metastasizing tumours of the rat and interferes with the steps not only of haematogenous, but also of lymphogenous metastasis.

We develop a highly specific antibody-dye conjugate for optical imaging of peripheral lymph nodes. The contrast agent consists of the monoclonal antibody recognizing endothelial ligands for the lymphocyte homing receptor L-selectin, MECA-79, and a near-infrared (near-IR) fluorescent indotricarbocyanine dye. The targeting and biodistribution behavior of MECA-79 is studied after radio-iodination and intravenous injection into mice demonstrating specific uptake in lymph nodes and accumulation in high endothelial venules (HEV). After conjugation of MECA-79 with indotricarbocyanine dye, the fluorescence imaging properties of the MECA-79 dye conjugate are examined by intravenous injection in nude mice and laser-induced fluorescence whole-body imaging in vivo. The MECA-79 antibody-dye conjugate accumulates in peripheral lymph nodes, whereas an isotype antibody-dye conjugate does not. Specific lymph node near-IR fluorescent signals become detectable within minutes after injection, and stable imaging persists for more than 24 h. The results demonstrate that vascular targeting of endothelial expression of glyocproteins is feasible to visualize the accumulation of near-IR fluorescent MECA-79 in lymph nodes, making this technology potentially useful to characterize processes of inflammation.